This invention relates to signal transduction via G-protein-coupled receptors (hereinafter GPCRs).
More specifically, this invention relates to compositions and methods for screening modulators of GPCRs. The compositions and methods involve mutated G proteins that can be used to shift signal transduction of certain classes of GPCRs to an alternative effector pathway that can be measured by high throughput screening techniques. Generation of such mutants therefore facilitates large-scale rapid screening for candidate GPCR modulators having diagnostic and/or therapeutic benefit.
The interaction between extracellular ligands and cell surface receptors is of central importance in the sensitivity and responsiveness of eukaryotic cells to exogenous signals and other environmental stimuli. Over the past decades, numerous cell surface receptors have been discovered, characterized and cloned. Among them, G-protein-coupled receptors (GPCRS) plays a pivotal role in a wide range of physiological responses.
G-protein-coupled receptors form a large family of related proteins. Members of each subfamily are coupled to different subsets of downstream signal transduction components that transduce and amplify distinct arrays of intracellular signals. In particular, GPCRs generally comprise a characteristic group of seven transmembrane spanning regions, and are further characterized by their ability to interact with heteromultimeric G-protein complexes, which are generally trimeric complexes comprised of xcex1, xcex2 and xcex3 subunits. Typically, activation of a GPCR by an agonist (i.e. a positive modulator such as a specific ligand) causes the GPCR to interact with and activate a particular class of G protein. In the activated G protein, GDP bound to the a subunit is replaced with GTP followed by the dissociation of xcex1GTP from the xcex2xcex3 dimer. The activated G protein subunits (xcex1GTP or free xcex2xcex3) are then able to modulate downstream signal transduction events via one or more xe2x80x9ceffectorxe2x80x9d molecules (typically via effector enzymes and/or the modulation of ion channels. Many effectors can be regulated by the xcex1 subunit and independently co-regulated (either positively or negatively) by the xcex2xcex3 units (see, e.g., E. J. Neer, Cell 80: 249-257, 1995).
At least four subfamilies of G protein xcex1 subunits have been identified to date, namely xcex1q, xcex1s, xcex1i and xcex112 (as illustrated in FIG. 6), and multiple xcex2 and xcex3 subunits have been found. More than half of GPCRs appear to couple to the heterotrimeric G proteins Gs or Gi in which the xcex1 subunit is xcex1s or xcex1i. Both Gs and Gi signal through the cyclic AMP (cAMP) pathway. In particular, Gs stimulates adenylyl cyclase, resulting in an increase in intracellular cAMP concentration, whereas Gi inhibits adenylyl cyclase, causing a decrease in intracellular cAMP level. A smaller number of GPCRs activate a phospholipase C (PLC) pathway by coupling with a distinct group of heterotrimeric G proteins, Gq in which the xcex1 subunit is xcex1q. Phospholipase C hydrolyzes phosphoinositides to generate two classes of well-characterized second messengers, namely, diacylglycerol and inositol phosphates. Diacylglycerol activates certain protein kinase Cs (PKCs) and certain inositol phosphates stimulate the mobilization of calcium from intracellular stores.
Among the physiological responses involving signal transduction via GPCRs are the dilation/constriction of blood vessels, bronchi and organs within the gastrointestinal tract, the modulation of endocrine secretions, and the control of heart rate. Indeed, in mammals it is believed that more than 1000 genes encode GPCRs of one type or another. Of those, several hundred GPCRs are likely to be involved in various disease processes, and thus are potential diagnostic and/or therapeutic targets. While GPCR modulators (including agonists and antagonists for example) have a great potential as diagnostic/therapeutic agents, the search for such agents is often both time consuming and labor intensive.
While some screening assays have been described, many assays are either inconvenient to perform on a large scale, or their uses are limited to only a subset of GPCRs. For example, while calcium mobilization (a downstream event associated with Gq activation) is amenable to high throughput screening using robotic assays, the majority of GPCRs are not coupled to Gq. Conversely, while many GPCRs are coupled to G proteins affecting changes in cyclic AMP (i.e. Gs or Gi as noted above), changes in cAMP level are not particularly amenable to high throughput or robotic screening assays.
There thus remains a considerable need for compositions and methods that can be used to facilitate the large-scale and rapid screening of GPCR modulatory agents, particularly agents that modulate GPCRs that are associated with alterations in intracellular cAMP (which collectively represent the majority of known GPCRs).
The present invention provides compositions and methods for identifying agents that modulate G-protein-coupled receptors, particularly agents that modulate non-Gq-coupled receptors (such as Gi- or Gs-coupled receptors), using high throughput screening methods based on Gq signaling.
In essence, the compositions and methods of the present invention effectively couple non-q upstream signaling events (such as those initiated by ligand binding to Gi- or Gs-coupled receptors) to q-type downstream signaling, such as calcium mobilization, that is readily amenable to high throughput screening techniques. The effective shift in signal transduction pathways greatly facilitates screening for GPCR modulatory compounds which would be of major diagnostic and therapeutic potential.